The biosynthetic pathways and control mechanisms of the tubercle bacillus are the broad subject of investigation at the Tuberculosis Research Laboratory. In the present project request, we propose to determine the exact site and mechanism of action of isoniazid on the inhibition in the synthesis of long-chain fatty acids (a possible precursor of mycolic acids) in the H37Ra strain of Mycobacterium tuberculosis and to determine directly if this inhibition is the primary mode of mycobactericidal action of isoniazid. Our long-range objective will be to elucidate the pathway to the synthesis of mycolic acids in the tubercle bacillus. We propose to accomplish the following; (a) To isolate, purify and characterize the C22-C56 fatty acids that are assumed to be precursors of mycolic acids. (b) To locate the exact site of action of isoniazid inhibition in the synthesis of long-chain fatty acids. (c) To isolate the active cell-free enzyme systems from M. tuberculosis that catalyze the synthesis of the C22-C56 acids (desaturase, elongation enzyme and cyclopropane synthetase). (d) To confirm the site of action of isoniazid with the isolated enzyme system and to study the nature of this inhibition. (e) To isolate the C27-C34 and C34-C40 fatty acids and add them back to the culture of M. tuberculosis to show the reversal of isoniazid effect. (f) Attempt to show the synthesis of mycolic acids in a cell-free system by utilizing a combination of the substrates meroacid CoA, meroaldehyde, trehalose-hexacosanoate and hexacosanoyl-CoA. (g) Completely characterize the two important mycolate-containing free lipids. (h) To isolate and test the appropriate cell-free enzyme system from an isoniazid resistant mutant for sensitivity to isoniazid.